Numerous fields of application require determining a multiplicity of analytes in a sample of a possibly complex composition and nature, for example in diagnostic methods for determining the state of health of an individual or in pharmaceutical research and development for determining the influence of an organism and the complex mode of action thereof by supplying biologically active compounds.
While known analytical separation methods are usually optimized in order to fractionate a very large number of compounds present in a given sample according to a predefined physicochemical parameter such as, for example, the molecular weight or the quotient of molecular charge and mass, in as short a time as possible, bioaffinity assay methods are based on using in each case one biological or biochemical or synthetic recognition element of very high specificity, also referred to as “binding partner” or “specific binding partner” hereinbelow, in order to recognize and bind the corresponding (individual) analyte in a sample of a complex composition in a highly selective manner. Detection of a multiplicity of different compounds thus requires the use of a corresponding number of different specific recognition elements.
An assay method based on bioaffinity reactions may be carried out both in a homogeneous solution and on the surface of a solid support (“substrate”). Depending on the specific design of the method, the latter requires, after binding of the analytes to the corresponding recognition elements and, where appropriate, further detection substances and also, where appropriate, between various method steps, in each case washing steps in order to separate the produced complexes of said recognition elements and the analytes to be detected and also, where appropriate, further detection substances from the rest of the sample and the optionally employed additional reagents.
To determine a multiplicity of analytes or study a multiplicity of samples, methods comprising detection of different analytes in discrete sample receptacles or “wells” of “microtiter plates” are widespread, especially in industrial analytical laboratories. Most widespread here are plates with a grid of 8×12 wells over an area of typically approx. 8 cm×12 cm, with a volume of a few hundred microliters being required to fill a single well. However, it would be desirable in numerous applications to determine a plurality of analytes in a single sample receptacle at the same time, using as small a sample volume as possible. WO 84/01031, U.S. Pat. No. 5,807,755, U.S. Pat. No. 5,837,551 and U.S. Pat. No. 5,432,099 propose immobilization of analyte-specific recognition elements in the form of a small “spots” as discrete measurement areas, some of which are well below 1 mm2, on a shared substrate in order to be able to determine the concentration of the analyte by binding only a small portion of analyte molecules present in a manner which depends only on the incubation time but which is, in the absence of a continuous flow, essentially independent of the absolute sample volume. A multiplicity of such “spots” as measurement areas in a two-dimensional arrangement on a shared substrate form a “microarray”.
Methods for simultaneously detecting a multiplicity of different nucleic acids in a sample with the aid of corresponding complementary nucleic acids immobilized on a substrate in discrete, spatially separated measurement areas as recognition elements are now relatively widespread. For example, arrays of oligonucleotides as recognition elements, which are based on simple glass or microscope slides as substrates and which have a very high feature density (density of measurement areas on a shared solid support), have been disclosed. U.S. Pat. No. 5,445,934 (Affymax Technologies), for example, describes and claims arrays of oligonucleotides having a density of more than 1000 features per square centimeter.
Recently, descriptions of arrays and assay methods of a similar kind carried out therewith for simultaneously determining a multiplicity of proteins, for example in U.S. Pat. No. 6,365,418 B1, have become more frequent.
The simplest form of immobilizing the binding partners for analyte detection consists of physical adsorption, for example due to hydrophobic interactions between the binding partners and the substrate. However, the extent of these interactions can be modified greatly due to the composition of the medium and its physicochemical properties such as, for example, polarity and ionic strength. The adhesive capability of the binding partners after purely adsorptive immobilization on the surface is often insufficient, in particular if various reagents are added sequentially in a multi-step assay.
Preference is therefore often given to immobilizing the binding partners on an adhesion-promoting layer applied to the substrate. A multiplicity of materials are known as being suitable for preparing said adhesion-promoting layer, for example non-functionalized or functionalized silanes, epoxides, functionalized, charged or polar polymers and “self-assembled passive or functionalized mono- or polylayers”, alkyl phosphates and alkyl phosphonates, multifunctional block copolymers such as, for example, poly(L)lysine/polyethylene glycols.
For example, WO 00/65352 describes coatings of bioanalytical sensor platforms or implants for medical applications as substrates with graft copolymers as adhesion-promoting layer, having a polyionic main chain (electrostatically) binding, for example, to a substrate and “non-interactive” (adsorption-resistant) side chains.
In order to minimize unspecific binding of analytes or their detection substances or other binding partners, in particular in the (uncovered) areas between the measurement areas (spots) for analyte detection, generated by way of locally addressed application, preference is frequently given to “passivating” these areas. For this purpose, compounds which are “chemically neutral”, i.e. non-binding, with respect to the analytes or with respect to their detection substances or other binding partners are applied to the substrates between the spatially separated measurement areas.
Said components which are “chemically neutral” with respect to the analytes or their detection substances or other binding partners, i.e. which do not bind these (also referred to as “passivation compounds” hereinbelow), are known to be able to be selected from the groups consisting of albumins, in particular bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, heterologous antibodies or antibodies empirically unspecific to the analyte(s) to be detected (in particular for immunoassays), detergents—such as, for example, Tween 20—, fragmented natural or synthetic DNA which does not hybridize with polynucleotides to be analysed, such as, for example, an extract of herring or salmon sperm (in particular for polynucleotide hybridization assays), or else uncharged but hydrophilic polymers such as, for example, polyethylene glycols or dextrans.
In order to be able to generate the likely quantifiable data from the binding signals, for example with the aid of fluorescence detection, of various measurement areas (spots) of a microarray, it is necessary to ensure a uniform surface density and binding activity of immobilized binding partners in measurement areas to be compared with one another. An essential precondition for meeting this requirement is high spatial homogeneity of the adhesion-promoting layer on which the discrete measurement areas (spots) are generated. Similar requirements also exist for the spatial homogeneity of the applied passivation layer to ensure a uniform, very low signal background between the designated measurement areas (spots).
Various methods can be employed for applying the adhesion-promoting layer to the substrate, depending on the molecular nature of the components of the adhesion-promoting layer to be generated and of course the thermal and chemical stability of the substrates to be coated. Silanizations may be carried out, for example, both in gas and liquid phases, for example with the aid of dipping methods. While the coating processes in the gas phase, in sufficiently large reaction vessels (compared to the size of the substrates to be coated), usually result in good homogeneity of the deposited layer, layers deposited from the liquid phase often have large spatial inhomogeneities, for example run tracks after application of dipping methods.
Since depositions from the gas phase usually require elevated process temperatures, the step of applying passivation layers usually takes place from the liquid phase, after applying the recognition elements for analyte detection which in most cases are not heat-resistant. The passivation step typically utilizes a dipping method. This involves dropping the substrate into a vessel filled with a solution of the compounds which are “chemically neutral” with respect to the analytes or their detection substances or other binding partners, i.e. which do not bind these (“passivating solution”), in order to wet the entire surface of the substrate as quickly as possible and simultaneously with the passivating solution. Subsequently, the substrate is left in the passivating solution (“incubated”) for a defined period of time for enabling the compounds employed for surface passivation to be adsorbed to the substrate surface.
An advantage of this conventional method is the fact that it can be carried out without any further aids and does not require any special demands on the abilities of the laboratory personnel.
A disadvantageous property of this method, however, is a relatively high risk of “smudging” of spots at the moment when the substrate is immersed, by passivating solution flowing past the substrate surface. In the process, material desorbs from the discrete measurement areas (immobilized specific binding partners) and is washed away and can be adsorbed again in the surrounding area in the direction of the relative direction of flow of the passivating solution (based on the substrate surface) in areas which are not yet completely covered with passivating compounds.
The extent of “smudging” of spots depends inter alia on the surface density of the immobilized specific binding partners in the discrete measurement areas and on the composition of the passivating solution, in particular on the solubility of the specific binding partners in said passivating solution. In the case of a high feature density, i.e. in the case of a short distance between neighboring spots, the “smudging” effect may greatly impair or even rule out quantitative analysis of the assay signals from an array of measurement areas, due to the resulting inhomogeneous distribution of background signals from the areas between said discrete measurement areas. This unwanted effect may result in a meaningful analysis of the assay signals being no longer possible, in particular if immobilized material is transported even from one spot to a neighboring spot. Another disadvantage of this method is the inherent need for relatively large volumes of passivating solution and relatively high costs associated therewith.
The described “smudging” effect is known to be prevented by the use of spraying methods, for example with the aid of atomizers. This involves applying the passivating solution in the form of small liquid droplets to the substrate surface until a continuous liquid film has formed on said surface. The substrate surface is then incubated in a saturated atmosphere of the liquid vapor (i.e. at 100% atmospheric humidity in the case of an aqueous passivating solution) within a predefined period of time, again in order to thereby enable the compounds employed for surface passivation to be adsorbed to the substrate surface. Run tracks are avoided by storing the substrates horizontally (with respect to the substrate surface to be coated) during said passivation process.
The spots can substantially be prevented from “smudging” by carrying out this process correctly. Another advantage is the amount of passivating solution needed, which is typically reduced by a factor of 10 compared to the dipping method described above.
However, a difficulty inherent to the method is the required uniform wetting and quite accurate metering of the amount of liquid applied, which are required for producing a homogeneous liquid film on the substrate surface, thereby putting increased demands on the operators. For example, “smudging” of the spots can again occur in the event of passivating solution being applied in excess. The international patent application WO 01/57254, for example, describes a modular system based on this coating method for producing microarrays with nucleic acids, proteins or other chemical or biological compounds as specific binding partners immobilized in discrete measurement areas.
Despite clear advantages in comparison with the dipping method, the results of the spraying method are likewise not optimal. Due to the fact that the droplets are expelled via a nozzle or an atomizer, said droplets possess a more or less strong momentum directed toward the surface to be coated at the moment when they hit said surface. This is associated with the risk of said droplets spattering when hitting the surface to give even smaller droplets, so that the edges of the measurement areas (spots) to be generated are usually not generated in a well-defined manner. Moreover, spraying methods usually generate relatively large droplets with a large variation in droplet size.